K17反义寡核苷酸对角质形成细胞增殖和K17表达的影响

目的研究脂质体介导角蛋白17(K17)反义寡核苷酸对培养人角质形成细胞增殖和K17表达的影响。方法利用脂质体将人工合成的正义、反义及错配K17寡核苷酸基因片段导入体外培养的人角质形成细胞系HaCaT,应用MTT法检测其对HaCaT细胞增殖的影响,以逆转录聚合酶链反应(RT-PCR)检测K17mRNA水平的变化,并以蛋白质印迹法、免疫荧光细胞化学结合激光扫描共聚焦显微镜检测K17蛋白水平的改变。结果脂质体介导的K17反义寡核苷酸转染HaCaT细胞后,细胞增殖受到明显抑制,同时细胞中K17mRNA和蛋白的表达明显下降,而正义寡核苷酸组、错义寡核苷酸组及空白对照组均无明显变化。结论应用反义技术封闭K17基因,可以阻遏角蛋白K17基因和蛋白的表达,抑制角质形成细胞的体外生长和增殖能力。     

皮肤科学基础

20 0 4 196 7　反义寡核苷酸阻遏角蛋白 14的表达 /陈玉欣 (四军大西京医院皮肤科 )… / /中华皮肤科学杂志 .-2 0 0 4 ,37( 4) .- 2 2 4～ 2 2 6人表皮角质形成细胞原代培养 ,利用脂质体将人工合成的正义、反义及错配 K14寡核苷酸基因片段导入角质形成细胞 ,应用流式细胞仪、逆转录聚合酶链反应 ( RT- PCR)和免疫组化 ( SABC)法检测反义寡核苷酸对角质形成细胞的细胞周期、K14基因和蛋白表达的影响。结果显示 ,应用反义技术封闭K14基因 ,可以阻遏角蛋白K14基因和蛋白的表达 ,并可以抑制体外培养的角质形成细胞的增殖。提示以 K14为靶…     

角蛋白K14反义寡核苷酸对角质形成细胞增殖的影响

目的：研究角蛋白K14反义寡核苷酸(ASODN)对人角质形成细胞(KC)体外增殖活性的影响。方法：利用脂质体将人工合成的正义、反义及错配K14寡核苷酸基因片段导人体外培养的角质形成细胞，应用细胞生长抑制实验、透射电镜(TEM)和流式细胞仪(FCM)检测ASODN对角质形成细胞的增生、超微结构改变和细胞增殖周期的影响。结果：脂质体介导的K14反义寡核苷酸组KC增殖活性受到明显抑制；电镜下可见KC增殖活性受到抑制的改变，角蛋白合成明显减少；流式细胞仪检测见细胞周期发生明显改变，G1期细胞百分率上升，S期细胞百分率下降。而正义寡核苷酸组、错义寡核苷酸组及空白对照组均无此改变。结论：K14反义寡核苷酸可抑制KC体外增殖活性，应用反义寡核苷酸技术封闭K14基因，有望为银屑病的基因治疗提供新的思路。     

皮肤科学基础

20042639　角蛋白K14反义寡核苷酸对角质形成细胞增殖的影响/陈玉欣(四军大西京医院全军皮肤性病中心)…//临床皮肤科杂志.-2004,33(5).-263～265采用脂质体将人工合成的正义、反义及错配寡核苷酸基因片段导入体外培养的角质形成细胞,应用细胞生长抑制实验、透射电镜和流式细胞仪分别检测反义寡核苷酸对角质形成细胞的增殖、超微结构和细胞增殖周期的影响。结果显示,脂质体介导的K14反义寡核苷酸对角质形成细胞的增殖活性有明显抑制作用;电镜下见角质形成细胞中角蛋白合成明显减少;流式细胞仪检测见细胞周期发生明显改变,G1期细胞百分率上…     

反义蛋白激酶Cζ对角质形成细胞增殖的影响

目的 研究蛋白激酶C(PKC)ζ亚类在角质形成细胞增殖中的作用.方法 采用基因转染的方法将PKCζ基因导入角质形成细胞株Colo16中,初步研究了表达反义PKCζ对Colo16角质形成细胞增殖的影响.结果 表达反义PKCζ的角质形成细胞形态发生改变,生长速率明显减慢,被阻抑在G1期.结论 反义PKCζ抑制Colo16角质形成细胞的增殖.     

角蛋白17反义寡核苷酸对银屑病SCID小鼠模型的作用

目的 建立重症联合免疫缺陷(SCID)小鼠银屑病动物模型,观察角蛋白17(K17)反义寡核苷酸(ASODN)对银屑病动物模型的作用.方法 将斑块状银屑病患者的皮损移植于SCID小鼠,构建银屑病SCID小鼠模型.然后分别以脂质体介导的K17 ASODN、正义寡核苷酸(SODN)及单纯乳剂基质作用于皮损,大体及H-E染色观察皮损改变,RT-PCR以及免疫组化检测K17的变化.结果 成功构建了SCID小鼠银屑病动物模型:与SODN组及单纯乳剂基质对照组相比,K17 ASODN治疗组皮损炎症恢复明显加快,组织学评分显著降低(P<0.01),K17 mRNA水平以及蛋白表达水平也均有不同程度降低.结论 K17 ASODN能够显著改善SCID鼠移植模型的银屑病病理改变,有望成为一种新的治疗措施.     

抗角蛋白自身抗体对角质形成细胞凋亡的影响

目的 探讨抗角蛋白自身抗体(AK auto Ab)对角质形成细胞凋亡的影响。方法 以不同浓度AK auto Ab作用体外培养人角质形成细胞后,用光镜及透射电镜观察细胞形态变化、用流式细胞仪分析细胞周期变化以及提取角质形成细胞DNA作电泳特征分析。结果 AK auto Ab作用后培养的角质形成细胞光镜、电镜下形态发生凋亡特有的改变,出现核固缩、染色质凝聚,并形成凋亡小体;细胞周期分析图中出现凋亡峰;DNA电泳呈有一定间隔的梯状条带。结论 AK auto Ab对角质形成细胞的凋亡具有诱导作用。     

靶向角蛋白17基因的小干涉RNA对角质形成细胞增生与凋亡的影响

目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。     

天然抗角蛋白自身抗体抑制角质形成细胞及鳞状细胞癌细胞增殖的作用

目的 探索天然抗角蛋白自身抗体抑制角质形成细胞及鳞状细胞癌细胞增殖的作用机理。方法 经体外培养人角质形成细胞及鳞状细胞癌细胞,作用于不同浓度的天然抗角蛋白自身抗体,四甲基偶氮唑盐法检测活性,免疫组化检测增殖及凋亡相关蛋白变化。结果 天然抗角蛋白自身抗体对二者的增殖有着明显的抑制作用,且呈一定的浓度-效应关系。对二者P53、C-myc的表达具有促进作用,而对增殖细胞核抗原的表达则具有抑制作用。结论 天然抗角蛋白自身抗体对角质形成细胞及鳞状细胞癌细胞增殖的抑制作用,可能系通过调节细胞中增殖与凋亡相关蛋白的表达来实现。     

抗角蛋白单克隆抗体5G5对培养角质形成细胞增殖的影响

目的 研究抗角蛋白单克隆抗体5G5在无血清培养的角质形成细胞中的免疫学活性,观察其对角质形成细胞增殖的影响。方法 免疫组化观察5G5对角质形成细胞的反应性;从培养的角质形成细胞中提取角蛋白,免疫印迹法检测5G5所识别的角蛋白区带;四甲基偶氮唑蓝比色法(MTT法)测定5G5对细胞增殖的影响。结果 5G5仅识别角质形成细胞中相对分子质量为50000的角蛋白区带,呈现为一条很强的染色带,而未与其它分子质量的角蛋白反应;免疫组化染色明显呈胞浆阳性;该单克隆抗体明显抑制培养的角质形成细胞增殖,并且存在剂量依赖关系。结论 特异性抗相对分子质量为50000角蛋白的抗体可能是角蛋白抗体家族中影响角质形成细胞增殖的有效成分。     

存活素反义寡核苷酸对角质形成细胞增殖和凋亡的影响

目的探讨存活素反义寡核苷酸对角质形成细胞增殖和凋亡的影响。方法以脂质体介导存活素反义寡核苷酸转染体外培养的角质形成细胞。采用MTT方法观察存活素反义寡核苷酸对角质形成细胞生长曲线的影响;采用RT-PCR方法观察存活素反义寡核苷酸对角质形成细胞存活素表达水平的影响;采用流式细胞仪检测存活素反义寡核苷酸对角质形成细胞细胞周期和凋亡的影响。结果存活素反义寡核苷酸作用于角质形成细胞后,细胞增殖受到明显抑制,存活素mRNA表达水平明显下降,细胞出现明显的凋亡现象。结论存活素反义寡核苷核酸能够抑制角质形成细胞增殖,促进角质形成细胞凋亡。提示存活素可能会成为一个新的治疗银屑病的靶分子。     

COX-2反义寡核苷酸对Colo-16细胞生长及c-myc基因表达的影响

目的探讨脂质体介导环氧合酶(COX)-2反义寡核苷酸(antisense oligodeoxynucleotide,AsODN)对皮肤鳞状细胞癌细胞株Colo-16细胞生长及对c-myc表达的影响。方法将COX-2无义(Nonsense oligodeoxynucleotide,NsODN)、反义寡核苷酸用脂质体分别导入Colo-16细胞中。四甲基偶氮唑蓝(MTT)法观察COX-2反义寡核苷酸对Colo-16细胞体生长曲线的影响;Hoechst33258荧光染色法观察细胞凋亡的形态学变化;蛋白免疫印迹法(Western blot)检测c-myc蛋白表达的变化。结果COX-2反义寡核苷酸作用于Colo-16细胞后,细胞增殖能力受到抑制,荧光染色可见细胞核染色质边集、固缩、核碎裂的凋亡形态学改变;c-myc表达明显下调。结论COX-2反义寡核苷核酸能够抑制Colo-16细胞体外增殖,诱导Colo-16细胞凋亡,并能显著下调c-myc表达。     

Flow cytometric and microscopic characterization of the uptake and distribution of phosphorothioate oligonucleotides in human keratinocytes

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a ‘bright’ population of highly fluorescent small cells and a ‘dim’ population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocytes was dependent on oligonucleotide concentration, incubation time and temperature. This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be considered when specific oligonucleotides are used against skin disease-specific genes. Received: 14 May 1997     

CLIC4, an intracellular chloride channel protein, is a novel molecular target for cancer therapy

Chloride intracellular channel (CLIC)4 is a p53- and tumor necrosis factor alpha (TNFalpha)-regulated chloride channel protein that is localized to the mitochondria and cytoplasm of mouse and human keratinocytes. CLIC4 protein increases in differentiating keratinocytes and in keratinocytes exposed to DNA-damaging agents and metabolic inhibitors. Increasing CLIC4 levels by transduction of recombinant CLIC4 causes apoptosis. CLIC4 translocates to the nucleus under a variety of conditions of cell stress, and nuclear CLIC4 is associated with cell cycle arrest and accelerated apoptosis. Reduction of CLIC4 and several other CLIC family members by expressing a doxycycline-regulated CLIC4 antisense also causes apoptosis in squamous cancer cell lines. Expressing antisense CLIC4 in tumors derived from transplanting these cells into nude mice inhibits tumor growth, increases tumor apoptosis, and reduces tumor cell proliferation. Co-administration of TNFalpha intraperitoneally enhances the tumor-inhibitory influence of CLIC4 antisense expression. Together, these results suggest that CLIC4 is important for keratinocyte viability and may be a novel target for anti-cancer therapy.     

Kinesin participates in melanosomal movement along melanocyte dendrites

Movement of melanosomes along melanocyte dendrites is necessary for the transfer of melanin pigment from melanocytes to basal and suprabasal keratinocytes, an event critical to epidermal photoprotection and maintenance of normal skin color. Recent murine data suggest that in melanocyte dendrites the microtubule-associated melanosome movement is bidirectional and that actin-associated myosin V secures the peripheral melanosomes, preparing them to be transferred to surrounding keratinocytes. We now report that human melanocytes express high levels of kinesin, a molecule that participates in microtubule-associated transport of organelles in other cell types, and that ultrastructurally kinesin molecules are closely associated with melanosomes. To determine whether kinesin participates in melanosomal transport, cultured melanocytes were treated with sense or antisense oligonucleotides complementary to kinesin heavy chain sequences. Antisense oligonucleotides decreased kinesin protein levels and inhibited the bidirectional movement of the melanosomes, promoting their backward movement. Furthermore, guinea pigs were exposed to ultraviolet B irradiation, known to enhance transport of melanosomes from melanocytes to epidermal keratinocytes, and then were treated with kinesin sense or antisense oligonucleotides. The areas that were treated with kinesin antisense oligonucleotides showed significantly less pigmentation clinically and histologically than control (sense) oligonucleotide-treated areas. As observed ultrastructurally, in antisense-treated areas melanosomes remained in melanocyte dendrites but over several days were not transferred to the surrounding keratinocytes. Our study supports a major role for kinesin in microtubule-associated anterograde melanosomal transport in human melanocyte dendrites.     

A novel gene expressed in human keratinocytes with long-term in vitro growth potential is required for cell growth

The herpes simplex virus large subunit of ribonucleotide reductase differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it contains a unique domain that codes for a distinct serine-threonine protein kinase that activates the Ras/MEK/MAPK mitogenic pathway and is required for virus growth. Previous studies suggested that ribonucleotide reductase protein kinase was co-opted from a cellular gene. Cellular genes similar to ribonucleotide reductase protein kinase were not cloned, however, and their function is unknown. Here we report that a novel gene (H11) that codes for a protein similar to herpes simplex virus 2 ribonucleotide reductase protein kinase, is expressed in skin tissues, cultured keratinocytes, and the keratinocyte cell line A431. The protein is phosphorylated and it associates with the plasma membrane. H11 is expressed in keratinocytes with long-term in vitro growth potential and is coexpressed with high levels of adhesion molecules involved in signal transduction, such as beta1 integrin. Antisense oligonucleotides that inhibit H11 expression inhibit DNA synthesis and keratinocyte proliferation, suggesting that H11 expression is required for cell growth.